The present invention provides immunotoxins comprising fusion proteins of non-mammalian cytotoxic RNAses and immunoglobulins and immunoglobulin fragments.
Ranpirnase is a member of a class of non-mammalian ribonucleases (RNAses) that show promise as cyotoxic agents for the treatment of disease. The cytotoxicity of these RNAses has been attributed to a mechanism that involves initial binding to cell surface receptors and internalization to the cell cytosol, with concomitant degradation of ribosomal RNA and inhibition of cellular protein synthesis. See Wu et al, J. Biol. Chem., 268:10686-10693 (1993). The RNAse activity is resistant to mammalian RNAse inhibitors, which may explain the enhanced cytotoxicity that is observed compared to the mammalian enzymes. Id. Ranpirnase is the prototypic member of this family of RNAses, and can be purified from Rana pipiens oocytes and early embryos. Ranpirnase has a molecular weight of 12,000 and causes potent inhibition of protein synthesis in the rabbit reticulocyte lysate (IC50 10−11 M) and when microinjected into Xenopus oocytes (IC50 10−10 M). Unlike other members of the RNase A superfamily, these cytotoxic ribonucleases do not degrade oocyte rRNA. Lin et al., Biochem Biophys Res Commun. 204:156-62 (1994). These molecules contain an obligate N-terminal pyroglutamyl residue that forms part of the phosphate binding pocket of the enzyme, and that is essential for RNAse and anti-tumor activity.
Animal toxicology studies show that ranpirnase displays a predictable, dose-dependent and reversible toxicity in both rats (dose range 0.01-0.02 mg/kg) and dogs (0.005-0.15 mg/kg). Mice inoculated with the aggressive M109 Madison lung carcinoma and treated with both daily and weekly schedule of intraperitoneally administered ranpirnase, showed significantly prolonged survival. Most striking results were seen in a group of mice treated with a weekly schedule of ranpirnase in which six of eighteen animals survived long-term and were apparently cured of cancer. Mikulski et al. J Natl Cancer Inst. 82:151-3 (1990).
Native ranpirnase has been shown in clinical trials to have anti-tumor activity against a variety of solid tumors. In this regard it has been used both alone and combined with other anti-tumor agents such as tamoxifen, e.g., when treating patients with pancreatic cancer. When used as an anti-tumor agent, these cytotoxic RNAses can be conjugated to a marker to permit targeting to a specific cell type.
In a Phase I study, patients suffering from a variety of relapsing and resistant tumors were treated intravenously on a weekly basis with ranpirnase (dosage range 60-960 μg/m2). Side effects observed included flushing, myalgias, transient dizziness, and decreased appetite in general. The observed toxicities, including the dose-limiting renal toxicity manifested by increasing proteinuria, peripheral edema, azotemia, a decreased creatinine clearance, as well as fatigue, were dose-dependent and reversible, which is in agreement with the animal toxicology studies. No clinical manifestation of a true immunological sensitization was evident, even after repeated weekly intravenous-doses of ranpirnase. The maximum tolerated dose, mainly due to renal toxicity, was found to be 960 μg/m2. There were also some objective responses in non-small cell lung, esophageal, and colorectal carcinomas. See Mikulski et al., Int J Oncol 3:57-64, (1993); Mikulski et al. J Clin Oncol. 20:274-81 (2002). Nevertheless, ranpirnase was well-tolerated by animals and the majority of human patients tested, demonstrated a consistent and reversible clinical toxicity pattern, and did not induce most of the toxicities associated with most conventional chemotherapeutic agents, such as myelosuppression and alopecia.
WO 97/31116 discloses a recombinant ribonuclease having (a) an N-terminal methionine followed by an amino acid other than glutamic acid, (b) a cysteine at positions 26, 40, 58, 84, 95 and 110, a lysine at position 41, and a histidine at position 119 (as aligned for maximum correspondence with bovine RNAse A), and a native ranpirnase-derived amino acid sequence. Recombinant cytotoxic RNAses, including RNAse variants, have, however, been prepared in bacteria by a multi-step process that requires additional steps to remove the N-terminal formylmethione residue inserted by the bacteria and to generate the obligate N-terminal pyroglutamyl residue. Nevertheless, production in bacteria precludes the preparation of glycosylated cytotoxic RNAse-containing fusion proteins. Accordingly, it would be advantageous to produce a recombinant cytotoxic RNAse fusion protein in eukaryotic cells where the cytotoxic RNAse retains the cytotoxic properties of ranpirnase purified from amphibian sources, but that has fewer, or no, undesirable immune responses in humans. However, it also would be expected that expression of cytotoxic RNAse in eukaryotic cells would result in cell death due to the cytotoxic activity of the RNAse.
It is apparent, therefore, that improved methods of preparing cytotoxic RNAse-containing fusion proteins, including glycosylated RNAse fusion proteins, are greatly to be desired. It also is apparent that glycosylated cytotoxic RNAse fusion proteins themselves are highly desirable molecules.